1. Technical Field
The present invention relates to a method and reagents for a fluorescence polarization immunoassay (FPIA) procedure for quantitating 3-methoxy-4-hydroxyphenylglycol (MHPG) levels in biological fluids such as urine, serum or plasma, which assay is characterized by the use of a novel stabilizing matrix for the fluorescent tracers used therein and by an improved specimen preparation method utilizing a magnesium silicate resin for reduction of specimen background and interfering metabolites.
2. Background Art
MHPG is the principal metabolite of the neurotransmitter norepinephrine in the central nervous system. Pretreatment MHPG levels in urine or blood provide the physician with information that aids in discriminating among subgroups of the major depressive disorders and that may aid in predicting patient response to specific antidepressants. Several studies have shown that differences in MHPG metabolism provide the basis for tentative identification of three biochemically discrete subgroups of the major depressive disorders. The first subgroup comprises bipolar manic depression, schizoaffective depression and some cases of unipolar endogenous depression. Patients in this subgroup excrete low levels of MHPG (x&lt;1.9 mg/24 hour) and may have low norepinephrine output. Patients in the second subgroup, presenting symptoms of endogenous depression, excrete intermediate levels of MHPG (1.9 mg/24 hours.ltoreq.x.ltoreq.2.5 mg/24 hours) and may have normal norepinephrine metabolism with abnormalities in other neurochemical systems. The third subgroup includes patients with symptoms of unipolar endogenous depression. These patients are characterized by high MHPG levels (x&gt;2.5 mg/24 hour), may have high norepinephrine output and, in the case of a somewhat discrete second tier subgroup, may also hypersecrete urinary cortisol.
First and second subgroup patients show a positive response to an amphetamine challenge, while third subgroup patients generally yield a negative response. Urinary and blood MHPG levels are predictive of the effectiveness of antidepressant drug therapy only for the unipolar endogenous depressive patients of the first subgroup. MHPG levels in the second and third subgroups have not been shown to have predictive value as to the success of drug treatment, and response to antidepressant therapy varies widely. Urinary and blood MHPG levels in patients with bipolar manic depression vary with alterations in affective state. Generally, these patients excrete lower levels of MHPG during depressive episodes, with higher MHPG levels during manic or hypomanic episodes than during periods of clinical remission.
Paper chromatography, spectrophotometric or fluorometric measurement, gas chromatography with flame ionization detection or electron capture detection, and radioimmunoassay have been described in the literature for MHPG determination in urine. Methods involving gas chromatography with mass spectrometry and high performance liquid chromatography with electrochemical or fluorescence detectors have also proven suitable for MHPG measurement.
In general, competitive binding immunoassays have provided a preferable alternative to chemical methods such as gas chromatography and high pressure liquid chromatography, which require sample extraction procedures and lengthy assay times. This is true of the fluorescence polarization immunoassay of the present invention, which combines the specificity of an immunoassay with the speed and convenience of a homogeneous method to offer a precise and reliable procedure for monitoring MHPG levels in urine or blood.
In a competitive binding immunoassay, the biological substance being measured (usually referred to as the "ligand") competes with a labeled reagent (the "ligand analog" or "tracer") for a limited number of receptor binding sites on antibodies specific to the ligand and ligand analog. The concentration of ligand in the sample determines the amount of ligand analog which binds to the antibody. The amount of ligand analog that will bind is inversely proportional to the concentration of ligand in the sample, because the ligand and the ligand analog each bind to the antibody in proportion to their respective concentrations.
MHPG is found both free and as sulfated and glucuronidated conjugates in urine. Only free MHPG will bind to antibody specific for MHPG. When MHPG from a urine sample or from a standard solution is equilibrated with MHPG tracer, the amount of MHPG tracer bound to the antiserum will be inversely proportional to the amount of free MHPG in the sample or standard. Thus, sulfated or glucuronidated conjugates must first be deconjugated to free MHPG if the competitive binding immunoassay is to provide an accurate quantitation of total MHPG present.
Fluorescence polarization is one method that may be utilized to measure the amount of tracer-antibody conjugate produced in a competitive binding immunoassay. Fluorescence polarization techniques are based on the principle that a fluorescent labeled compound, when excited by plane polarized light, will emit fluorescence having a degree of polarization inversely related to its rate of rotation. Accordingly, when a tracer-antibody conjugate having a fluorescent label is excited with plane polarized light, the light remains highly polarized because the fluorophore is constrained from rotating between the time that light is absorbed and the time that it is emitted. In contrast, when an unbound tracer is excited by plane polarized light, its rotation is much faster than that of the corresponding tracer-antibody conjugate; as a result, the light emitted from the unbound tracer molecules is depolarized.
Fluorescence polarization immunoassay techniques are well known in the art; to date, however, the use of such techniques for the determination of urinary MHPG levels has not been successfully attempted. The present invention offers an advance in the art in that highly accurate determinations of the amount of MHPG in urine are obtainable with increased convenience, at decreased expense, without the disadvantages heretofore imposed by the various available assay systems.